Work

Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving

Year: 2010

Type: article

Abstract: The large diversity of cells that comprise the human immune system requires methods that can resolve the individual contributions of specific subsets to an immunological response. Microengraving is process that uses a dense, elastomeric array of microwells to generate microarrays of proteins secreted from large numbers of individual live cells (approximately 10(4)-10(5) cells/assay). In this paper, we describe an approach based on this technology to quantify the rates of secretion from single immune cells. Numerical simulations of the microengraving process indicated an operating regime between 30 min-4 h that permits quantitative analysis of the rates of secretion. Through experimental validation, we demonstrate that microengraving can provide quantitative measurements of both the frequencies and the distribution in rates of secretion for up to four cytokines simultaneously released from individual viable primary immune cells. The experimental limits of detection ranged from 0.5 to 4 molecules/s for IL-6, IL-17, IFNgamma, IL-2, and TNFalpha. These multidimensional measures resolve the number and intensities of responses by cells exposed to stimuli with greater sensitivity than single-parameter assays for cytokine release. We show that cells from different donors exhibit distinct responses based on both the frequency and magnitude of cytokine secretion when stimulated under different activating conditions. Primary T cells with specific profiles of secretion can also be recovered after microengraving for subsequent expansion in vitro. These examples demonstrate the utility of quantitative, multidimensional profiles of single cells for analyzing the diversity and dynamics of immune responses in vitro and for identifying rare cells from clinical samples.

Source: Lab on a Chip

DOI: 10.1039/b926849a

Authors: Qing Han, Elizabeth M. Bradshaw, Björn Nilsson, David A. Hafler, J. Christopher Love

Institutions: Massachusetts Institute of Technology, Brigham and Women's Hospital, Harvard University, Broad Institute, Yale University

Language: English

Topic: Advanced Biosensing Techniques and Applications

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: green

Funders: Deshpande Center for Technological Innovation, Massachusetts Institute of Technology, National Institute of Allergy and Infectious Diseases